Modified Hodge test: A useful and the low-cost phenotypic method for detection of carbapenemase producers in Enterobacteriaceae members.

BACKGROUND: The global spread of antimicrobial resistance has acquired greater significance in the public health perspective. Drug resistance has posed a threat for the management of various hospital-acquired infections (HAI). For bacteria producing extended spectrum ß lactamase, carbapenems are the drug of choice. However, treatment failures are still a cause of concern due to carbapenemase producers. AIM: Various phenotypic and genotypic methods are available for the detection of carbapenemase producers. Studies thus far have mostly concentrated on comparing various methods for detection of carbapenemase producers. We used low-cost and the easily performed modified Hodge test (MHT) for detecting the carbapenemase producers in Enterobacteriaceae members isolated from various clinical specimens.
MATERIAL AND METHODS: The study included 1072 clinical isolates of Enterobacteriaceae collected in India between April 2008 and February 2010. MHT was performed on all the isolates in accordance with CDC and CLSI guidelines.
RESULTS: The carbapenemase activity was detected in 35.9% (385/1072) of the isolates. Klebsiella spp. 28.7% (80/278), Citrobacter spp. 20.4% (25/122), 11.3% (38/334) in E. coli, 20.3% (45/221) in Enterobacter spp., and 16.2% (9/117) in Proteus spp. revealed variable resistance activities against carbapenems.
CONCLUSION: Enterobacteriaceae members are among the most common and easily transferable bacterial species responsible for severe HAI. This study revealed a high percentage of Enterobacteriaceae clinical isolates producing carbapenemases in India. Detection of such bacteria, formulating hospital antibiogram, and monitoring the usage of antimicrobial drugs is recommended.

INTRODUCTION

In view of the increasing incidence of infections caused by multidrug resistant (MDR) microbes, there are very limited options to treat such infections.[1] Few antimicrobial drugs have, in the past two decades, been introduced in the market, compared to the remarkably growing number of both Gram-positive and Gram-negative MDR bacteria.[2] Several members of the Enterobacteriaceae family of bacteria are normally present as harmless human gut flora. However, these bacteria are the leading cause for a wide range of opportunistic infections.[3] Although the carbapenemase activity has mainly been detected in Pseudomonas and Acinetobacter clinical isolates, recent studies have demonstrated the emergence of carbapenem resistance among Enterobacteriaceae members in different geographical regions, which is of great concern as these bacteria are easily transmissible among patients, leading to hospital acquired infections (HAI), but can also spread into the community, resulting in community-acquired cases.[4] Carbapenem resistance in Enterobacteriaceae may be due to various reasons that include hyper production of the Amp C beta lactamase, loss of porins, production of metallo-beta-lactamases (MBL) and production of K pneumonia carbapenemases. The most important carbapenemase determinants responsible for resistance or reduced susceptibility to carbapenem group of antibiotics in Enterobacteriaceae members include class A KPC 1-3, Class D (OXA-48, OXA-181), IMP, VIM, NDM, NMC-A, SME1-3, IMI-1, GES-2, SHV, and SFC.[5] Multidrug resistant gram negative bacteria due to production of beta lactamase, metallo-beta-lactamases, and carbapenemases are difficult to treat. In view of the alarming increase in the appearance of Carbapenemase-producing bacteria in the clinical isolates a standard testing method should be followed for detection of carbapenemase producing bacteria. The carbapenemase detection methods include the modified Hodge test (MHT), the double disk test (DDST), blood agar combined disk (BA-CD) assay, PCR amplification, and DNA sequencing.[6] Bacteria susceptible to the carbapenem group of antibiotics also need to be tested as some may still have the enzyme. The study aimed to investigate the carbapenemase activity in Enterobacteriaceae members, using the modified Hodge test.

MATERIALS AND METHODS

The study, performed at Apollo Health City, Hyderabad, India, included 1072 consecutive clinical isolates of Enterobacteriaceae, collected between April 2008 and Feb 2010. The MHT was performed on all the isolates irrespective of their susceptibility pattern to carbapenems (Imipenem, Meropenem, and Etrapenem), in accordance with guidelines of the Centers for Disease Control (CDC) and Clinical and Laboratory Standards Institute (CLSI) (http://www.cdc.gov/; http://www.clsi.org/).[7] Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 1706 and Pseudomonas aeriginosa ATCC 27853 were used as controls.

A lawn culture was prepared on Mueller Hinton agar (MHA) using an overnight culture suspension of E. coli (ATCC 25922) adjusted to 0.5 McFarland's standards. The plate was left for 15 min for drying and then a disc of 10 μg meropenem was applied at the centre of plate. The isolates under study were streaked from the edge of the disk to the periphery of the plate. Four isolates were tested in each plate. After an overnight incubation at 37° C, the clover leaf like appearance between the test streaks near the disk was taken as positive for carbapenemase production.

RESULTS

The isolates were identified as E. coli (n = 334), Klebsiella spp. (n = 278), Enterobacter spp. (n = 221), Proteus spp. (n = 117), and Citrobacter spp. (n = 122). Overall, the carbapenemase activity was detected in 35.9% (385/1072) of the isolates. The activity varied between 28.7% (80/278) in Klebsiella spp., 20.4% (25/122) in Citrobacter spp., 11.3% (38/334) in E. coli, 20.3% (45/221) in Enterobacter spp., and 16.2% (9/117) in Proteus spp. MHT positive and negative is shown in Figures 1 and 2.

Figure 1

Clover leaf appearance of carbapenemase positive strains

Figure 2

Arrow depicting carbapenemase negative strain

DISCUSSION

The increased occurrence of MDR and resistance to carbapenems in Enterobacteriaceae has been a cause of concern to public health. This study detected a high percentage 35.9% (385/1072) of the potential carbapenemase activity among a collection of Enterobacteriaceae isolates obtained from various clinical specimens in India. Among the different Enterobacteriaceae members tested in the present study, Klebsiella spp. showed the highest percentage of carbapenem resistance (~30%), whereas Proteus spp. and Citrobacter spp revealed comparatively low carbapenem resistance of (~17%) and (~12%), respectively. A recent study from South India done as a part of antimicrobial surveillance program (SENTRY) that tested 39 Enterobacteriaceae isolates collected between 2006 and 2007 that showed reduced susceptibility to carbapenem antibiotics revealed 26 (66.6%) were found MHT positive.[8] Deshpande et al. in their recent study tested 24 carbapenem resistant Enterobacteriaceae members and found 22 (91.6%) were MHT positive and later confirmed by PCR.[9] A study from Greece included ~117 ESBL negative Enterobacteriaceae members that revealed a MHT positivity of 41.8%.[10] The high carbapenemase positivity in this study can be attributed to the low-level hydrolysis of carbapenems by ESBLs of the CTX-m type.[11] The limitation of this study is not comparing the results with a molecular method as the main aim of the study was to present data on the carbapenemase producing Enterobacteriaceae members that can be useful in developing strategies to control the spread of such bacteria as we still have no guidelines to treat infections caused by carbapenemase producing bacteria.[12] Previous reports have confirmed that carbapenem susceptible isolates showed the presence of carbapenemase gene by PCR indicating that clinical resistance may not be detected in Enterobacteriaceae members and that laboratories should routinely check for carbapenemase production among clinical isolates by possible phenotypic or genotypic methods.[11] Several phenotypic methods for carbapenemase detection are available, of which the MHT is recommended both by CDC and the CLSI.[1314] Other tests including the combination disc tests have been in use by many laboratories.[15] Studies have compared MHT and combination disc tests and found variable results. Amongst them few studies have raised concern over the false positivity in the MHT test.[16] Few other works have proved combined disk synergy tests as more effective in detecting the carbapenemase activity. The carbapenemases of the OXA, KPC, IMP, and VIM types are clinically important enzymes. They are all encoded on mobile genetic elements, located on plasmids or chromosomes, and are frequently isolated from patients suffering from antibiotic resistant infections.[5] Studies have come to a contrasting conclusions about use of MHT, one finding it as inadequate in detecting the metallo-beta-lactamases and others proving that MHT produces false positive carbapenemases.[16] An Indian study showed MHT is not preferred for carbapenemases in nonfermenting gram negative bacteria and recommended the use of both EDTA-meropenem and the EDTA-ceftazidime combination test.[17] This study results clearly demonstrate the presence of the carbapenemase activity in high percentage of Enterobacteriaceae members detected by the MHT that has proven to be easily done in any tertiary care setting with minimal infrastructure and is cost effective. Routine testing of all clinical isolates for possible carbapenemase activity may result in availability of data on such isolates as only few studies have been done in this regard. Confirmation of the resistance mechanism is not required in the public health perspective. Studies must be encouraged to assess the risk factors for infections with carbapenemase producing bacteria. Effective antibiotic policy, infection control programs combined with good medical practices can help in confronting the menace of antibiotic resistance.